In the past, antiviral and antibacterial research has focused mainly on viral and bacterial targets. Due to continued growth of drug resistant organisms the search for effective and differentiated antiviral and antibacterial therapies continues. Development of immune-potentiating agent is one of the strategies being pursued to identify new anti-infective agents. An adjuvant is an agent administered to potentiate the immune response to an antigen and/or modulate it towards a desired immune response. An endogenous adjuvant is a compound or molecule naturally occurring within the cell or tissue that likewise enhances an immune response by stimulating innate immunity, thus possessing the capacity to potentiate an effect of some triggering event or agent. Endogenous adjuvants play a central role in alerting the immune system to potential danger and promote response to infection, transplantation, tumor, and autoimmunity.
Some endogenous adjuvants are known to promote CD8+ T-cell immune responses which are important in combating infections associated with pathogens, such as viruses and bacteria as well as controlling the tumor growth (Rock et al, in Springer Seminars in Immunopathology (2005) 26:231-246).
PNP deficient patients have demonstrated significantly high levels of plasma nucleosides, inosine, deoxyinosine, guanosine and deoxyguanosine compared to normal healthy subjects (Markert in Immunodeficiency Review (1991) 3:45-81) and also elevated levels of erythrocyte deoxyguanosine triphosphate (dGTP) and nicotinamide adenine dinucleotide (NAD). Plasma deoxyguanosine (only nucleoside measured in the clinic) and intracellular dGTP was elevated in patients treated with PNP inhibitor (Bantia and Kilpatrick in Current Opinions in Drug Discovery & Development (2004) 7: 243-247). Deoxyguanosine was also elevated in mouse plasma after treatment with PNP inhibitor (Bantia et al. in International Immuno-pharmacology (2001) 1:1199-1210 and (2010) 10:784-790).
A major source of nucleoside pools comes from the breakdown of RNA and DNA during normal cell turnover, cellular injury or cell death due to infection. Normally the nucleosides deoxyguanosine, inosine, deoxyinosine, and guanosine are present at very low levels in the plasma because PNP is an extremely efficient catalyst and rapidly breaks down inosine and deoxyinosine to hypoxanthine, and, guanosine and deoxyguanosine to guanine and sugar 1-phosphate. In the presence of PNP inhibitor or due to a PNP deficiency, however, these nucleosides, guanosine, inosine, deoxyinosine, and deoxyguanosine, are elevated.
Guanosine analogs like isatoribine (7-thia, 8-oxoguanosine), loxorabine (7-allyl, 8-oxo guanosine) and others have shown to be immuno-potentiators and have demonstrated antiviral, antibacterial and anticancer effects in animal models (Smee et al. in Antimicrobial Agents and Chemotherapy (September 1989) 1487-1492; Stewart et al. in J. Interferon Cytokine Research (2012) 32(1):46-51; also in Poult Science (2012) 91(4):1038-1042; Pope et al. in Cell Immunol. (1995) 162(2):333-339).
ANA773, an oral pro-drug of isatoribine, and has demonstrated induction of endogenous interferon-a (IFN-a) of multiple subtypes in healthy volunteers. ANA773 in clinical trials of chronically HCV infected patients demonstrated dose dependent reduction in HCV RNA (Bergmann et al. in Aliment Pharmacol Ther (2011) 34:443-453; International patent number WO2005025583A2).
In-vitro studies with these guanosine analogs have shown activation of immune cells like dendritic, natural killer cell to produce ifn-gamma which is mediated through Toll-Like Receptor 7 (TLR7). Toil-like receptors (TLRs) have been established as a family of pathogen recognition receptors (PRRs) that initiate the innate immune response. In addition to TLR.s there are other PRRs like retinoic acid inducible gene I (RIGI) like receptors (RLR), nucleotide binding oligomerization domain (NOD)-like receptors (NLR) as well as c-type lectin receptors (CLR). Stimulation of TLRs and PRRs directly or indirectly causes the release of multiple cytokines including type 1 and type 2 interferons, the induction of pathways and enzymes that destroy intracellular pathogens, the activation of a variety of cellular responses, and the priming of the adaptive response by activating immature dendritic cells and inducing their differentiation into professional antigen-presenting cells. At least eleven different TLR genes have been identified in humans. Through stimulation of innate immunity by activating TLR, isatoribine and other guanosine analogs does prevent or reverse otherwise lethal viral infections in various acute infection models in mice.
The Inosine analog methyl inosine monophosphate has also shown immune enhancing effects and demonstrated antiviral and antibacterial effects (Mishin et al. in Antiviral Research (2006) 71:64-68).
In addition to the accumulation of nucleosides in the presence of PNP inhibitor, deoxyguanosine is converted to dGTP in lymphocytes and erythrocytes and dGTP could stimulate the immune system through activation of PRRs in the presence of an antigen similar to what has been observed with ATP. Although the mechanism is not clear PNP deficient patients also demonstrate increase in the nucleotide, NAD levels. NAD may also serve as danger signal and activate the immune system (Haag et. Al., Purinergic Signalling (2007) 3:71-81)
Based on the role of PNP in purine catabolism, the present investigators hypothesize that inhibiting PNP may elevate nucleosides, inosine, deoxyinosine, guanosine and deoxyguanosine levels and nucleotides, NAD and dGTP in a subject as demonstrated in PNP deficient patients and PNP deficient mice (FIG. 1). Humans and mice treated with PNP inhibitor demonstrate significant increase in levels of plasma deoxyguanosine, the only nucleoside that has been measured.
Clearly it would be beneficial to provide methods for treating diseases which exploit the natural endogenous adjuvant response. Controlling levels of endogenous adjuvants provides a novel means to augment antiviral, antibacterial and anticancer treatments. Further, identification of endogenous adjuvants triggered in response to certain pathogens could provide novel exogenous adjuvants which may be administered to enhance an immune response and/or to potentiate the therapeutic efficacy of other antiviral, antibacterial and anticancer treatments.